The long-term goal of our program is to define the molecular, cellular and physiological mechanisms that regulate osteoblast growth and differentiation, within the context of skeletal development and bone formation in vivo. Major contributions of this program to understanding bone cell biology and pathology include (i) definition of distinct developmental stages and the principal transition points that together mediate establishment and maintenance of the osteoblast phenotype, (ii) characterization of the role of Runx2/Cbfa1 and other regulatory factors in the molecular mechanisms that regulate cell growth, as well as induction and physiological responsiveness of bone-phenotypic genes during osteoblast growth and differentiation, and (iii) development of genetic models (e.g., using transgenic and gene knock-out mice) that provide the basis for bone tissue specific gene therapy. In the current funding period, we have identified BMP2 target genes that activate the Runx2 gene and play a central role in mediating osteoblast growth and differentiation. We hypothesize that analysis of the physiological regulation of the bone-related Runx2 P1 promoter will lead us to combinatorial mechanisms that integrate developmental signaling pathways to control the onset and progression of osteogenesis. Based on our preliminary results, we further postulate that BMP2, Wnt signaling and (homeodomain) proteins together contribute both to induction of Runx2 gene expression, and to functional activity of Runx2 and downstream target genes. This hypothesis will be addressed experimentally by characterizing (i) developmental regulation of the bone related Runx2 P1 gene in vivo in our Runx2 P1-LacZ knock-in mouse;(ii) Runx2 as an integrator of cell signaling through selective protein-protein interactions during distinct stages of osteoblast differentiation, and (iii) Runx2 and BMP2 dependent signaling components that mediate osteoblast commitment of mesenchymal progenitor cells. Thus, the proposed studies will advance understanding of molecular, cellular and genetic mechanisms that initiate and support complete development of the osteoblast phenotype, as well as provide the basis for clinical strategies to treat a broad range of bone-related disorders.